Differential vacuum chamber for directed transport of a substance

ABSTRACT

In a vacuum chamber, which has at least two vacuum regions, substances, especially fluids, are transported in a directed manner from one region into the other region by the application of a suitable vacuum. As a result, it is possibly by the use of filter supports or receiver supports to transport a fluid automatically through corresponding filter supports during a plasmid preparation. The vacuum chamber is charged by means of a gripping robot that operates automatically and is switched by a suitable valve control means. By incorporating the vacuum chamber with its gripping robot and the control means into a pipetting robot system, automatic plasmid preparation, for example in accordance with the Qiagen protocol, is possible.

The present invention relates to a vacuum chamber and a vacuum systemusing the vacuum chamber for the directed transport of a substance,especially a fluid, and to its use in an apparatus for automatic plasmidpreparation.

The past few years have seen an increase in the scale of efforts toobtain the complete genetic information of entire organisms. Beginningwith the sequencing of a phage genome (bacteriophage T7: 38000 basepairs, bacteriophage λ: 48514 base pairs) and continuing by way of thegenome of Escherichia coil (4.2×10⁶ base pairs) to the yeastSaccharomyces cerevisiae (2.3×10⁷ base pairs) as the firstrepresentative of the eukaryotes, the number of base pairs to besequenced has increased almost 600-fold. In the meantime, the humangenotype with more than 3×10⁹ base pairs has become the goal of theseefforts in the “Human Genome Project”. The enormous quantities of DNA tobe sequenced are barely manageable by the means and personnel availableto laboratories hitherto. There is therefore a demand for newtechnologies that, for an acceptable financial outlay, are capable ofbringing about a considerable increase in the throughput of samples inthis research programme. Two mutually influencing strategies have cometo light in the course of current development: on the one hand theminiaturisation of laboratory sequences and on the other hand theunsupervised automation of well-established laboratory procedures.

The miniaturisation of laboratory sequences has given rise tominiaturised electrophoresis analysers in which the separation ofbiomolecules on the basis of their charge and size is utilised. Suchminiaturised electrophoresis analysers are obtained by means ofmicrostructures in electrophoresis chips. Also available areminiaturised PCR machines, wherein during the polymerase chain reaction(polymerase chain reaction=PCR) DNA fragments up to 6 kilobases in sizeare amplified. Also known are miniaturised sample arrays andminiaturised detection systems. Miniaturised elements such as thosedescribed above can be combined to form larger units, so that a completeminiaturised laboratory unit is obtained.

On the other hand, for the automation of a laboratory it is notabsolutely necessary to miniaturise routine procedures. It is likewisepossible to design a robot system that completely or partially replacesthe manual tasks of a human being in order to achieve an increase insample throughput. The following manual tasks are typical of alaboratory preparation (with particular emphasis on plasmid preparationas a preliminary to PCR sequencing) and need to be carried out bysuitable robots:

pipetting

transport of used material and of chemicals

suction of fluids through filters, membranes, permeable solids or thelike

PCR reaction.

Current pipetting robots locate standard laboratory material on a worksurface at defined positions and thus enable tested laboratory protocolsto be set up. For example, using auxiliary robots it is possible formicrotitre plates, pipette tips or reservoirs for buffer solutions etc.to be installed on such machines and, after use, removed from theworkstation again. Thus, all the necessary pipetting steps preliminaryto a PCR or a plasmid preparation can be executed in order that theproduct of that pipetting operation can then be introduced into asuitable machine for further preparation using a gripping robot.

The polymerase chain reaction (PCR) amplifies a DNA segment when it isenclosed between two defined primer sites. If equal amounts of primersare used, double-stranded DNA copies are produced by the PCR, whereas ifone primer is used in excess then, in accordance with that excess,single-stranded copies of the amplified DNA are obtained. Bothsingle-stranded and double-stranded DNA can be used for sequencing. Insequencing-intensive projects the DNA fragments to be analysed arecloned into plasmids which are then in the first instance present in adefined matrix of bacterial colonies (Escherichia Coli Blue) growing onagar. The subsequent taking up of the colonies from the matrix intoculture tubes can also be automated. Over an incubation period (37° C.)lasting about 12 hours the living bacterial clones then yield sufficientmaterial to obtain in a preparation the plasmid copies necessary forsequencing. Obtaining such purified plasmids for sequencing is achieved,for example, by the QIAWELL 96 ultraplasmid purification procedure. Suchplasmid preparation procedures include filtering operations in which afluid has to be transported in a directed manner from one filter into atleast one second filter and either also passes through that filter or issimply collected in a controlled manner.

The problem underlying the invention is therefore to provide anapparatus in which the directed movement of substances can be carriedout automatically.

The present invention relates to a vacuum chamber for the directedtransport of a substance, especially a fluid, there being installed inthe vacuum chamber a permeable means and a collecting means, so thatthere are defined at least two vacuum regions that can be establishedindependently of one another, namely a first vacuum region between thepermeable means and the collecting means and a second vacuum regionbetween the collecting means and the base of the vacuum chamber, and avacuum can be generated in the two vacuum regions independently of oneanother so that the substance, especially the fluid, can be sucked fromthe first permeable means into the collecting means. Fluids are here tobe understood as being gases, liquids, vapours and fumes.

The second means is preferably also permeable, so that by theapplication of a vacuum to the second region the fluid or liquid issucked through the second means into the second region. The vacuumchamber according to the invention will generally have exactly twovacuum regions, but more than two vacuum regions are possible, forexample when several filtrations are to be carried out one immediatelyafter another.

Furthermore, the permeable means are formed by filter supports having alarge number of filter elements, so that fluid can be transported in adefined manner from a particular filter element of the first filtersupport into a corresponding filter element of the second filter supportin turn through the latter into the second lower region of the vacuumchamber. The collecting means are likewise collector supports having adefined number of collecting elements. In the case of a collectorsupport, therefore, the substance, especially the fluid, is transportedthrough the first filter support into the collector support.

Moreover, the vacuum chamber consists of a cover and a lower part, thelower part of the vacuum chamber having a shoulder for receiving thelower filter support. In addition, recesses for the gripper of the robotare provided in the side walls of the chamber in order that the filterplates or filter supports can be inserted and removed automatically. Forthe exact receiving and guidance of the filter supports or the collectorsupport the lower part of the vacuum chamber has guide tabs havingcorrecting bevels. The guide tabs preferably have two different bevelangles, the first bevel angle being about 30° and the second bevel anglebeing about from 0° to 2°. Furthermore, the guide edges of the wall withwhich the filter supports come into contact on insertion can bebevelled. Preferably the cover has a bevelled guide edge so that whenthe cover is put in place it is centred using the guide edge of thecover. The edge bevel angle is preferably 30°. The cover also has in thewall region recesses for the robot gripper and a supporting surface forthe upper filter support.

The sealing material for the upper filter holder preferably has ahardness of about 20 Shore, the seal at the join between the cover andthe lower part being formed by a combination of an O-ring and aresilient sealing strip, the O-ring providing a seal of about 60 Shoreand the sealing strip of about 30 Shore. Sealing is also effected at thelower filter support using a rubber gasket having a hardness of 60Shore.

The upper part has corresponding receiving means for receiving the guidetabs so that the cover is centred on the lower part by means of theguide tabs.

In a preferred embodiment, the filter supports have N pipe-shapedindividual filters (N being especially 96) that are connected to form afilter support. The same applies to the collector support. In addition,there are mounted on the corresponding four corner pies of the twofilter supports, or of the filter support and the collector support,spacer sleeves which, in addition to their function of defining thefirst vacuum region, also effect the vertical correction ofmisplacements of the lower filter support by engaging in centring shaftsin the vacuum lower part. The spacer sleeves preferably have a partiallycylindrical shape in order to allow the vacuum to act on the cornerpies. In addition, the spacer sleeves can be bevelled so that anadditional centring of the filter supports is achieved during insertion.A filter support may be in one piece consisting of a large number offilter elements or it may be composed of a large number of individualfilter elements.

The length of the spacer sleeves is preferably so selected that theoutlet tips of the upper filter support are located inside the pipes ofthe lower filter support or collector support, so that a controlledtransport of the fluid through pipes or elements that correspond to oneanother is achieved. The outlet tips of the elements of the upper filtersupport are preferably located 1.5 mm inside the corresponding pipes ofthe filter elements or collecting elements of the corresponding lowersupport. As a result, contamination of non-corresponding elements isavoided.

Preferably the vacuum chamber and the spacer sleeves are manufacturedfrom plexiglass of a suitable thickness, which allows visual monitoring.For industrial production, the vacuum chamber may consist of a castplastics material, which allows economical manufacture.Injection-moulding processes and milling processes may also be used.

In the lower part of the vacuum chamber there are arranged a suctionshaft for the first vacuum region and a suction shaft for the secondvacuum region. Fluid passing through during a filtration procedure isremoved directly from the vacuum chamber through the suction shaft ofthe second vacuum region.

The present invention relates also to a vacuum system having at leastone vacuum pump and an electronically controlled valve for the lowerchamber region, an electronically controlled valve for the middlechamber region, a valve for breaking the creeping vacuum in the lowerchamber region and a vacuum trap arranged between the valves and theconnection to the lower region of the vacuum chamber for receiving thewaste volume. Each proportional valve may have its own controllingelectronics system which can be actuated by the control software via adecoding apparatus of a PC.

The invention relates also to an apparatus for automatic plasmidpreparation having a vacuum system for automating the directed transportof a substance, a pipetting robot and a gripping robot, wherein thegripping robot inserts the filter supports, after pipetting has beencarried out by the pipetting robot, into the vacuum chamber and closesthe cover and, after filtering, opens the chamber and removes the filtersupports and conveys them to a further processing step. Such anapparatus is preferably controlled by a computer. It is also possible towork with only one robot which assumes the gripping and the pipettingfunctions.

The apparatus also has a dryer for filter supports, because in somepreparation procedures the last preparation step is washing withalcohol, so that residual alcohol adhering to the last support has to beremoved.

Advantageously the vacuum chamber is part of a larger robot system whichcan be used for supporting all current preparation methods of molecularbiology processes. A modular design has therefore been created whichenables the apparatus components of the apparatus for automaticpreparation to be arranged to suit the particular problem being posed.Furthermore, the robot system used has no feedback, which means that novisual or other sensory monitoring of the current actual state ispossible. All the movable components of the system must therefore belocated in positions that are defined as exactly as possible. Whencomponents are moved by a robot arm, it is important that those movedcomponents, when taken up by the robot again, are located at exactlydefined positions. The vacuum chamber is, in addition, removable fromthe system as a module so that other modular systems for other methodscan be inserted in its place. Therefore the position of all auxiliarysystems for the automated preparation is oriented on the pipettingrobot. In order, therefore, to be able to monitor the current positionof the gripping robot visually during the “learning phase” of thesystem, the vacuum chamber is advantageously manufactured fromplexiglass or some other transparent plastics material. It is readilypossible, however, to use a feedback robot system in which the feedbackis provided by sensors.

By the use of the vacuum it is possible to transport fluid, in two stepswell defined in time, from one filter into, for example, a second filterarranged below the first in order to be sucked through the second filterinto the lowermost region and disposed of.

The invention, especially the vacuum chamber with its at least twovacuum regions which are independent of one another, is not, however,restricted to the transport of fluid in the filtering phase of a plasmidpreparation. Other possible uses are the separation of mixtures, theinitiation of reactions, the establishment of adsorption processes bythe automation (expressed in general terms) of the directed transport ofa substance by the provision of at least two vacuum regions that can beestablished independently of one another. “Substance” is here to beunderstood as a single substance or a mixture of substances in the formof a fluid, gas or fumes. Furthermore, not only filter supports can beused as the permeable means but the use of, for example, an array ofminiaturised chromatography columns is likewise possible, so thattime-resolved transport of a substance can also be achieved.

Preferred embodiments of the invention are explained below withreference to the drawings.

FIG. 1 is a diagram showing the course of a plasmid purificationprocedure in accordance with a protocol of the Qiagen company;

FIG. 2 shows a perspective view of the lower part of the vacuum chamber;

FIG. 3 shows a perspective view of the cover of the vacuum chamber;

FIG. 4 shows an exploded view of the vacuum chamber according to theinvention;

FIG. 5 shows a centring tab used in the vacuum chamber;

FIG. 6 shows a spacer sleeve in longitudinal view, cross-sectional viewand side view;

FIG. 7 shows a cross-sectional view of the vacuum chamber with thefilter supports inserted and the cover not closed;

FIG. 8 shows a cross-section through the vacuum chamber with the filtersupports inserted and the cover in place;

FIG. 9 shows a diagrammatic view of the valve system;

FIG. 10 shows a diagrammatic view of the entire apparatus for automaticplasmid preparation;

FIG. 11 shows a diagrammatic view of the vacuum chamber electronicmodule, and

FIG. 1 is a diagram showing the course of a plasmid preparation orpurification, as used, for example, by the Qiagen company. In Step 1 theDNA fragments to be analysed are cloned into plasmids which are then inthe first instance present in a defined matrix of bacterial colonies(Escherichia Coli Blue) growing on agar. Over an incubation period at37° C. lasting about 12 hours the living bacterial clones then yieldsufficient material which is purified in accordance with the followingscheme. The pellets obtained after centrifugation are resuspended in thetest tubes in Step I and lysed. In Step II the samples are each pipettedinto a filter element of a filter support (QIAfilter 96 (yellow)). Inthe filter support of Step II, the cell walls etc. are retained in thefilter, while the DNA strands are flushed with the fluid into thecorresponding pipes of the next filter support in Step III. In thefilter elements of that second filter support (QIAWell 96 (white)) ofStep III, the DNA is adsorbed on the filters, while the filter fluidflows downwards. Using a buffer fluid, the DNA on the filter elements ofthe second filter support is washed and conveyed using an elution bufferinto a third filter support (QIAprep 96 (blue)) in Step IV. From therethe DNA or the plasmids are eluted into a support consisting ofcollecting elements in Step V. Those plasmids collected in theindividual test tubes can be conveyed to a PCR machine (not shown) inorder on the one hand to increase the number of copies of the DNA and/oron the other hand to carry out a PCR sequencing step.

FIG. 2 shows a perspective view of the lower part 1 of a vacuum chamberV which is used to transport fluid from an upper filter support (notshown) into a lower filter support (not shown) or collector support (notshown), By this means the various filtration steps II to V of theplasmid purification procedure, for example in accordance with theQiagen protocol of FIG. 1, are able to take place automatically, forexample using a robot, the fluids being sucked by means of a partialvacuum from an upper filter support into or through a lower filtersupport.

The lower part 1 of the vacuum chamber V comprises an inner chamber 2which is divided into a chamber base portion 3 and an upper portion 4,the division being made by a peripheral ledge 5 on which the lowerfilter support or receiver support is arranged. As a result of thisledge 5, the cross-section of the chamber base portion 3 is slightlysmaller than that of the upper portion 4. A groove 6 has been made bymeans of milling along the ledge 5 on the wall side, the significance ofwhich groove will be explained below. The lower part 1 has suctionshafts 7, 8, the suction shaft 7 serving to aspirate the lower chamberbase region 3, while the suction shaft 8 generates a vacuum in the uppervacuum region between the upper and lower filter supports. For theaccurate positioning of the filter supports F1, F2 in the vacuum chamberV there are provided in the upper region of the lower part 1 guide tabs9, 10, 11, 12 and 13 which project above the lower part 1. In addition,the upper end face 14 of the lower part 1 has a groove 15 for receivinga rubber seal 16 (not shown). The guide tabs 9 and 13 are provided withcorrecting bevels so that an enforced alignment of the filter supportF1, F2 is effected on insertion. In order to be able to insert thefilter supports F1, F2 there are provided in the lower part 1 of thevacuum chamber, in opposing side walls, grip recesses 17, 18 and 19 inwhich the gripping fingers of a robot hand engage. The lower part 1 alsohas on the guide edges of the chamber correcting bevels 20 which comeinto contact with the filter support or with spacer sleeves affixed tothe filter support. Locating bores 21 are provided for fixing theposition of the vacuum chamber V in the entire system. To allow thevacuum chamber V to be used with other filter supports or other filtersystems, the lower part 1 of the vacuum chamber additionally hasmilled-out centring shafts 22 and 23 in the corners.

FIG. 3 shows the cover 30 of the vacuum chamber V, viewed from below.For moving the cover 30, the cover likewise has grip recesses 31, 32 and33 for the robot grippers. The cover also has a 30° bevel at the edgefor receiving the upper filter support F1 (not shown). In the interiorthere is also arranged, as supporting surface for the filter support F1,a ledge 35, on which the filter support F1 is sealed by means of asuitable sealing material. The cover 30 also has a supporting surface 36which forms the counterpart to the sealing end face 14 of the lower part1. The cover 30 is provided with suitable recesses 37, 38, 39, 40 and 41in which the guide tabs 9 to 13 of the lower part 1 engage when thecover 30 is put in place and effect final alignment of the cover 30. Inthe preferred embodiment the cover 30 is open at the top (at the bottomin FIG. 3). This is necessitated, however, by the special use of thefilter supports which requires normal atmospheric pressure to be presenton the upper side of the upper filter support in order for the fluid tobe transported through the filter elements by the pressure differentialbetween the external air pressure and the upper vacuum region. For otheruses, where, for example, no vapour is to be allowed to escape into theenvironment, the cover 30 may be closed.

FIG. 4 shows an exploded view of the vacuum chamber V consisting of thelower part 1 and the cover 30, the lower part 1 and the cover 30 beingsealed with respect to one another by means of a seal 16 which rests inthe groove 15. The seal 16 does not press directly against the sealingsurface 36 of the cover 30, but instead there is located between them aperipheral sealing strip 42 having a hardness of 30 Shore. A seal 43,which seals the lower filter support (not shown) with respect to theledge 5 of the lower part 1, rests loosely on the ledge 5. The seal 43of the lower filter support F2 is preferably a rubber gasket having ahardness of 60 Shore. The upper filter support F1 is sealed with respectto the sealing surface 35 of the cover 30 by way of a sealing strip 44.The hardness of the seal 44 is about 20 Shore.

Also shown are bolts 45 with which the vacuum chamber V is fixed inposition by means of the bores 21.

FIG. 5 shows a plan view, a side view and a perspective view of a guidetab 9 to 13 used for centring. It will be seen that the guide tab 9 hastwo bevels 50, 51 which differ from one another, the first bevel havingan angle of about 30° and the second bevel having an angle of about from0 to 5°, preferably 2°. The dimensional data in FIG. 5 are in mm and thesize of a guide tab is 30×30×5 mm (height×width×thickness).

FIG. 6 shows the spacer sleeves 60 for the filter holders F1, F2, whichspacer sleeves 60 are pushed into the respective outer corner pipes of afilter support. The spacer sleeves 60 serve on the one hand to effectfinal alignment of the lower filter support F2 and to define a desiredspacing between the upper filter support F1 and the lower filter supportin order to create the upper vacuum region VO between the filtersupports for the purpose of sucking through the fluid from the upperfilter support into the lower filter support. If the filter supports F1,F2 were simply to be placed one above the other in the vacuum chamber Vit would be impossible to establish two separate vacuum regions in thechamber V. The outlet connections with outlet tips 82 of the filterpipes 83 of the upper filter support F1 fit tightly into the filterpipes 81 of the support F2 located below, so that a vacuum VOestablished in the intermediate region would not be able to suck outpreparation fluid located in the pipes of the upper filter holder F1. Itis therefore necessary to provide a sufficiently large gap, that is tosay upper vacuum region VO, between the two filter supports, F1, F2.Furthermore, it is absolutely necessary for reasons of preparationtechnology to prevent any of the fluid dripping down from being sprayedinto adjacent pipes of the lower filter system F2. Otherwise there wouldbe cross-contamination of neighbouring samples, which would render theresult of the preparation unusable. The spacing between the two filtersupports F1, F2 is therefore such that the outlet tips 82 of the upperfilter support F1 are located about 1.5 mm inside the filter elements 81of the pieces of the lower filter support F2. In order to ensure thatspacing and the definition of the first vacuum region, cylindricalspacer sleeves 60 of a defined radius are therefore placed into thecorner pipes, the spacer sleeves 60 being milled open, that is to sayprovided with a broad longitudinal slot 61, so that the corner pipesalso are acted upon sufficiently by the vacuum. In order to align thelower filter support F2, the end faces 62 of the spacer sleeves 60 arebevelled. In a preferred embodiment, the spacer sleeve 60 is 31.5 mmlong and has an outer diameter of 11.4 mm, the height of the partiallyopen cylinder being 7.8 mm. The internal diameter is fixed at 9.2 mm andthe end-face bevel is 0.5 mm×45°. This 45° bevelled portion makes thefirst mechanical contact with the corresponding guides on the lower part1. By virtue of the geometry of the spacer sleeves, the further downwardmovement of the gripper arm brings about the corrections necessary forthe filter supports F1, F2 to be received exactly. For the purpose ofeasy handling in laboratory operation, the spacer sleeves 60 are made ofa material that, on the one hand, has the flexible properties of springclips but, on the other hand, has sufficient resistance to chemicals andsufficient rigidity, so that the spacer sleeves 60 can be placedresiliently onto the four corner pipes of the filter supports F1, F2.1.5 mm thick sleeves 60 of plexiglass were therefore chosen. Forindustrial production they can be made economically from plexiglasstubes. The slot 61 necessary for positioning on the four corner pipes issufficiently large not to impede the establishment of the vacuum in thatarea. The spacer sleeves can also be arranged fixedly on the filtersupports, for example in an injection-moulding process or the like.

FIG. 7 shows the lower and upper filter supports, F1, F2 arranged oneabove the other in the lower part 1 of the vacuum chamber V, the outlettips 80 of the individual filter elements 81, which here are constructedin the form of pipes, of the lower filter support F2 projecting into thelower vacuum region VU of the vacuum chamber V. The outlet tips 82 ofthe filter elements 83 of the upper filter support F2 project into theupper pipe region of the filter elements 81 of the lower filter supportsF2. The upper vacuum region VO is defined by suitable selection of thespacer sleeves 60 that are positioned on the lower filter support F2.Also shown is the cover 30 before it is placed onto the lower part 1,the lower part 1 having in its sealing surface 14 a groove 15 with arubber seal 16 which effects a seal with respect to the seal 42 of thesealing surface 36 of the upper part 30. The upper filter support F1effects a seal with respect to the seal 44 of the upper part 30. In FIG.7, the two filter supports F1 and F2 can again be seen at the side. Thefilter supports F1 and F2, which consist of the filter elements 81, 83,may be constructed in one piece. It is also possible, however, for afilter support F1, F2 to be constructed as it were in modular form, forexample by assembling individual filter elements 81 or 83 by means ofsuitable connections. The filter supports F1, F2 also have side walls84. The lower filter support F2 is seated on the seal 43 of the ledge 5of the lower part 1 of the vacuum chamber and thus forms the lowervacuum region VU, which is vented or evacuated via the suction shaft 7.The upper vacuum VO is formed by the suction shaft 8. Furthermore, thegrip recesses 31, 32 and 17 for the robot grippers are shown by way ofexample. The grip recesses 31, 32, 17 are open towards the top by way ofa bevel in order to obtain better access for the robot. In their baseregion they have a groove so that the robot gripper can be braced in therecesses 31, 32, 17.

FIG. 8 shows the same situation as in FIG. 7 but now with the cover inplace, and it will be seen that the upper filter support F1 is sealedwith respect to the upper part 30 by the sealing surface 35 and acorresponding 44. Shown on the right-hand side in the drawing are thefilter supports F1, F2, which are arranged one above the other, theupper vacuum region VO being defined by means of the size of the spacersleeve 60.

The insertion of the filter supports F1, F2, shown diagrammatically inFIG. 1, into the vacuum chamber V and the components thereof inaccordance with FIGS. 2 to 6 will be explained below with reference toFIGS. 7 and 8.

When the filter supports F1, F2 are being introduced from a pipettingrobot PR (FIG. 10) into the vacuum chamber V by means of a grippingrobot GR (FIG. 10), slight changes in position may arise in the courseof their travel. If these changes are not eliminated, then in a robotsystem having no sensory feedback, as is the case here, there would soonbe a mechanical catastrophe. For that reason, an enforced mechanicalalignment is carried out by the vacuum chamber V itself during insertionof the filter supports F1, F2.

When the filter F2 that collects the fluid (lower filter) is insertedinto the vacuum chamber V, it is the guide tabs 9 to 13 that make thefirst mechanical contact with the lower part 1 of the vacuum chamber V.Because the filter support F2 may undergo slight changes in position inthe course of travelling from the pipetting station PR to the vacuumchamber V, the guide tabs 9, 10, 11, 12, 13 serve to bring the filtersupport F2 back into the required position during its insertion into thelower part 1 of the chamber V. The CRS 465 (gripping robot GR) used inthis preferred embodiment can travel towards predetermined co-ordinatesonly by means of a “spline function” calculated in the C500 controller.For that reason it is necessary always to specify movement sequencesthat are not stringent in respect of the spline function. The robotgripper arm with the filter support F2 therefore initially approaches atmoderate speed an approach position approximately 1 cm vertically abovethe final point of contact with the guide tabs 9 to 13. As the filtersupport F2 is slowly lowered, contact will be made with the bevels 50,51 of the guide tabs 9 to 13, misplacements being compensated forfirstly on the 30° bevels 50 and subsequently on the 2° inclinations 51of the surfaces aligned perpendicularly to the direction of movement viathe enforced downward movement of the gripper arm. After about 1 cm, thefilter support F2 if flanked by the five alignment faces that taperdownwards at an angle of 2° in such a manner that misplacements in thehorizontal region relative to the lower part 1 of the vacuum chamber 1are compensated for in every case. It is only then that the verticalalignment of the filter support F2 on the centring shafts 22, 23 of thechamber is effected. On the four outer corner pipes of the filtersupport there are mounted spacer sleeves 60 which, in addition to theirfunction of rendering the upper filter support F1 accessible to thevacuum in the upper region of the chamber V, here perform their secondfunction, namely the vertical correction for misplacements of the lowerfilter support F2. For this purpose, likewise 30° bevels 62 are locatedboth on the spacer sleeves 60 and on the centring shafts 22, 23 of thevacuum chamber lower part 1. After having travelled about 2 mm, thefilter support F2 is positioned by the downward movement of the robotarm in the correct position inside the lower part 1 of the vacuumchamber V. At the same time the gripping tool passes into the griprecesses 17, 18, 19 of the lower part 1. Approximately 1 mm above itsfinal deposition point, the filter support F2 is released by the gripperof the robot arm and thus comes to rest on the seal 43 which rests onthe ledge 5 of the lower part 1.

When the upper filter support F1 is installed by means of the robotgripper arm, its centring is effected in a similar manner by way of theguide tabs 9 to 13. The spacer sleeves 60 align the upper filter supportF1 parallel to the lower support F2 already located in the lower part 1.If, nevertheless, a misplacement of the upper filter holder F1 shouldoccur, it will be compensated for by the special construction of thevacuum chamber upper part 30. When, from a special part position, thecover 30 of the vacuum chamber V is placed by the gripper of thegripping robot onto the vacuum chamber lower part 1, the cover 30 makesits first contact with the guide tabs 9 to 13 of the lower part 1. Forthat purpose, in addition to having their 30° inner bevels, the latterhave also been given short 45° outer bevels. The cover 30 receives themin special recesses 37 to 41. The cover 30 is aligned using the recesses37 to 41 by way of the downward movement of the gripper. After about 0.5cm, the cover 30 has been mechanically aligned with the lower part 1 ofthe vacuum chamber V to such an extent that the second function of theupper part 30 can be carried out, namely the correction of an upperfilter holder F1 which may have been misplaced. For that purpose, thecover 30 is provided with 30° bevels 34 on the corresponding contactzones. The width of the bevel 34 is derived from the possiblemisplacement of the filter holder F1. Because the tolerances by way ofthe guide tabs 9 to 13 may be a maximum of 0.5 mm, the bevel 34 on thecover is about 0.5 mm wide on each side. This ensures sufficienttolerance in receiving the filter support F1. As a result of thedownward movement of the gripper, the filter holder F1 is, if necessary,moved positively into the suitable position by means of the bevels 34 ofthe cover 30.

The sealing of the assembled chamber V is effected in three places intotal. The lowermost zone is sealed by a rubber gasket having a hardnessof 60 Shore. The second zone is the join between the lower part 1 andthe cover 30 of the vacuum chamber V. It is formed by a combination ofan O-ring 16 (60 Shore) and a resilient (30 Shore) sealing strip 42. Thethird zones is sealed with respect to the inner cover region by theupper filter F1. The cover 30 therefore has two supporting surfaces 35,36 which are provided for mounting the sealing material. For thatpurpose they are roughened during the manufacturing process.

FIG. 9 shows the valve system necessary for operating the vacuumchamber. Starting from an oil pressure pump RD4, a vacuum hose isconnected at a Y-connection to two electric valves V1, V2, which servefor the controlled establishment of two vacuum regions in the chamber V,namely the upper vacuum region VO between the two filter supports F1, F2and the lower vacuum region VU for aspirating the fluid from the lowerfilter support F2. The valve V2, which is responsible for the uppervacuum region, is directly connected via a vacuum hose to thecorresponding connection 8 of the chamber V. The other valve V1, whichcontrols the evacuation of the base region, is connected via a vacuumhose to the vent pipe of a vacuum trap F. The lateral hose coupling ofthe trap F is connected by a hose connection to the connection 7 of thelower region of the vacuum chamber V. Using this arrangement, therefore,the water volume of about 500 ml can be removed from the system. Betweenthe vacuum trap F and the controlling valve V1 there is connected bymeans of a T-piece a vent valve V3 which plays an important role inmaintaining normal air pressure in the lower chamber region. The valveV3 makes a connection to normal air pressure.

The three potential valves V1, V2, V3 each have their own controllingelectronics system which can be actuated by the control software via adecoding apparatus of a PC. The decoding apparatus likewise assumes thecontrol of the oil pressure pump RD4, so that the control software isalso able to control its activity. The valves V1, V2, V3 can be simplegas valves without special coatings. Their opening and closing behaviourcan be altered by way of the controlling electronics system. Forexample, the two vacuum valves V1, V2, after receiving their actuationsignal, open to the preset extent linearly over a period of 2 seconds.This delayed opening prevents the vacuum chamber from being evacuatedtoo quickly by the connected oil pressure pump RD4. The control softwarefirst starts the oil pressure pump RD4 which then generates a partialvacuum in the entire region of supply to the valves V1, V2. After about5 seconds, depending upon the requirements of the preparation stepcurrently in progress, the control software sends the pulse for openingone of the two valves V1, V2. If that valve were to open too quickly,the resulting surge could damage the filter system or the vacuum chamberV. The vent valve V3, which is connected in parallel with the lowervacuum region of the chamber V, opens to its full extent linearly over aperiod of 0.1 second. It is then activated by the control software atpredetermined time intervals when the upper region of the vacuum chamberV is to be evacuated, but the lower it to have normal air pressure. Thisis the preparation step in which fluid is sucked from the pipes of theupper filter support F1 into the lower filter support F2. Here acreeping partial vacuum must be removed from the system as quickly andeffectively as possible. Because is can be assumed that in this case thepressure difference is small, the valve V3 is rapidly opened or closedto its full extent. A partial vacuum possibly building up in the baseregion of the chamber V is kept negligibly small by frequent venting inthis manner.

During the plasmid preparation a waste volume of about 500 ml is formedand must be conveyed out of the lower region of the chamber V. The wasteconsists of the chemicals necessary for the preparation and the cellresidues of the bacteria. The fluid therefore cannot be regarded asharmless from both its chemical and its biological nature and so it mustbe stored intermediately in a suitably secured container in order thatit can be disposed of in a controlled manner when the preparation workis complete. The vacuum chamber V has therefore been provided in itslower region with a suction site 7 which is connected via a removal linesystem to the vacuum trap VF. The vacuum present in the lower regionduring the preparation ensures that any fluid arising from the lowerfilter support F2 is immediately sucked into the vacuum trap VF, whichat the same time acts as an intermediate store. The control valve V1,which is responsible for the lower vacuum, is equipped with a brassclosure without special protective washers for reasons of cost. Becauseof the aggressive nature of the preparation fluids this control valve V1is arranged upstream of the vacuum trap VF. It is therefore alsopossible to select a vacuum trap VF of economical material (pressedglass). An oil pressure pump (model RD4, Vakubrand) having a suctionpower of 4.3 m³/h is used to generate the vacuum. If the oil pressurepump is activated, the vacuum builds up upstream of the valve V1 but notin the vacuum trap VF, which is under normal air pressure until thevalve V1, which is actuated by way of the electronic control means, isopened. The control means is so designed that the valve V1 opensproportionally over a period of 1 second, so that the vacuum does notbuild up so rapidly in the trap VF. The vacuum load on the trap VFtherefore exists only during the period in which the preparation fluidsare being moved through the filter materials. Because during that periodthe weakest point of the system is the 0.7 mm thick plastics dish of thefilter support F1, which is directly connected to normal air pressure,there is no risk of the vacuum trap's imploding at any time during thepreparation operation. Even if there should be a blockage of the filtermaterial in all the pipes of the filter support F1, F2, the vacuumchamber V is so constructed that the lower rubber seal 43 acts as asafety valve. The rubber seal 43 will be drawn out of its lateral bevelinto the chamber V and by means of the opening so produced the vacuumwill return to normal air pressure. Destruction of the filter support F2is therefore also ruled out. In order that the fluid to be sucked out isremoved as quickly as possible from the lower chamber region, thesuction opening 7 has been mounted directly on the base of the chamberV. The opening is connected directly to the suction connection 7 via abore.

In order to be able to compensate for slight material tolerances of thefilters F1, F2 and to achieve secure closure of the vacuum chamber V,the materials of the three seals have the following properties:

The sealing material of the lower chamber part 1 must be a material ofmoderate rigidity, in the present case the hardness is 60 Shore. Thisensures that during the cyclic breaking of the creeping vacuum from theupper chamber regions, the seal of the upper filter support F1 withrespect to the cover 30 is not broken.

The sealing material of the cover 30 must be very soft and resilient (20Shore). This ensures sufficient sealing of the cover 30 with respect tothe upper filter support F1 and with respect to the O-ring 16 of thelower chamber half 1. By virtue of its very resilient nature it is alsopossible to compensate for manufacturing tolerances of the filtersupport F1. The manufacturing tolerances of the filter supports F1, F2have a two-fold effect: on the one hand they affect the supportingheight of the lower filter support F2 and on the other hand they alsoaffect the sealing of the upper filter support F1 with respect to thecover 30.

The O-ring 16 of the lower part 1 seals the chamber V with respect tothe very soft sealing material 42 of the cover 30. The O-ring 16 has adiameter of 2 mm. As a result of its small supporting surface, aconsiderably smaller contact pressure of the cover 30 against the lowerpart 1 of the vacuum chamber V is necessary to provide an adequate seal.For example, the weight of the cover 30 together with the vacuum beingbuilt up in the interior of the chamber V is sufficient to close thecover 30 tightly with the filter supports F1, F2 it encloses. That pointis very important for an unsupervised preparation carried out by a robotsystem because a vacuum that does not build up correctly will bringabout the disruption of the entire plasmid preparation.

Furthermore, it is necessary to rely on directed aspiration of the fluidfrom the upper filter elements 83 into the filter supports or collectingcontainer F2 arranged below. The fluid must remain in the lower filtersupport F2 without being immediately sucked through into the base regionof the chamber V. When the vacuum develops in the upper region of thechamber V, then at the point at which fluid has not yet dripped into thelower filter support F2, a small amount of gas will rise upwards throughthe unwetted filter support F2. A slight vacuum will therefore be formedin the lower region of the chamber V. Once all the fluid has changedfrom the upper filter support F1 to the lower filter support, normalatmospheric pressure will become established in the cover region of thevacuum chamber V. As a result, the partial vacuum initially formed inthe base region of the chamber V will suck through some of the fluidthen located in the lower filter support F2 onto the base of the chamberV. In order to avoid this effect, the vent valve V3 is opened atintervals by the software control means. At the beginning of the ventingprocess, full opening of the vent valve V3 takes place after everysecond, then after about 10 seconds for every 5 seconds that haveelapsed, the opening speed of the valve V3 being at its maximum. Forsafety reasons, this cyclic venting of the lower vacuum region VU iskept constant while the upper vacuum region VO is connected to thevacuum. In the case of unsupervised robot preparations, a leakage in thelower region of the chamber V could result in a constant build-up of apartial vacuum. This can be compensated for by the cyclic ventilation ofthe lower region of the chamber V.

FIG. 10 shows a diagrammatic representation of the entire system forautomatic plasmid preparation. The system comprises a pipetting robot PR(BIOMEK 2000), the vacuum system having the pump RD4, the valves V1, V2,V3, the vacuum trap VF and the vacuum chamber V, a gripping robot GR(CRS 465), a PCR machine (PTC 225, MJ-Research), a fixed shelving systemRS for critical laboratory equipment (high salt buffer, acids etc.) anda carousel K (i.e. a rotatable shelving system for laboratoryequipment). The system also comprise a dryer T for the filter supportsF1, F2. For controlling the gripping robot GR, the latter is connectedto a Risc-Workstation RS (C500) which is operated by way of a serialinterface S (preferably an RS 232 interface) by a PC control computer PCwhich monitors the entire automatic plasmid preparation sequence. Thearrangement of the gripping robot GR is such that it has access to thefilter supports F1, F2 arranged on the pipetting robot PR, to the vacuumchamber V consisting of lower part 1 and cover 30, to the two shelvingsystems RS, K, to the dryer T and to the PCR machine PCR. The cover 30of the vacuum chamber V has its own deposition site. The preparationsequence of the gripping robot GR, which is equipped with athree-fingered hand, is as follows in accordance with the Qiagenprotocol: the gripping robot GR takes a filter support F2 for the lowerfilter from the carousel K and inserts it into the lower part 1 of thevacuum chamber V. It then takes from the pipetting robot PR the upperfilter support F1, the pipes (filter elements 83) of which have beenpipetted with the appropriate preparation fluid by the pipetting robotPR, and places it onto the lower filter F2 in the lower part 1 of thevacuum chamber V. The vacuum chamber cover 30 is then put in place andthe filtration, that is to say the transport of the fluid through thefilter supports F1, F2, is carried out by applying a suitable vacuum tothe upper or lower vacuum region. When the filtration is complete, thevacuum chamber V is opened automatically by the gripping robot GR andthe uppermost filter F1 is removed and disposed of. The lower filtersupport F2 is again conveyed into the pipetting robot PR and filled withthe appropriate preparation fluid, while the gripping robot GR takesfrom the carousel K a further filter support F2 to be used as lowerfilter support for the next filtration step and introduces it as lowerfilter support F2 into the lower part 1 of the vacuum chamber V. Thepipetted filter support is then taken from the pipetting robot PR andinserted as upper filter F1 into the lower part 1 and the vacuum chamberV is closed. When the transport of fluid is complete, the chamber V isopened again, the upper filter support F1 is disposed of and the lowerfilter support F2 is again conveyed into the pipetting robot PR for theintroduction of the next preparation fluid. If the next filtration stepis the last, the gripping robot GR takes a collector support from thecarousel and inserts it as lower support F2 into the lower part 1. Theappropriate pipetted filter support is then taken from the pipettingrobot PR and introduced as upper filter support F1 into the lower part 1and the vacuum chamber V is closed. In what is then the third filterstep, the preparation fluid containing pure DNA is sucked into thecollector support F2, and the filtration in accordance with the Qiagenprotocol is complete.

In order to control the entire system it is necessary to have a specialcontrol system and regulation means for each of the units. In otherwords, each of the modules of the entire system is provided with its owncontrol software. All the control operations are regulated within thecontrol software and do not require feedback to the commanding system.Windows NT is preferably selected as the operating system, because ithas stable pre-emptive multitasking which also supports phase-parallelsub-programs and it is possible to use interprocess communicationmethods.

Finally, FIG. 11 is a block diagram showing the actuation of the module70 of the vacuum chamber V. The module 70 comprises the sub-modules“control vacuum chamber” 71, “electronics system” 72, “valves” 73 and“oil pump RD4” 74, the module 70 being controlled by the module“commanding robot” 75, that is to say starts the actuation of the vacuumchamber. In the module 70, three valves V1, V2, V3 and an oil pressuresuction pump RD4 are controlled by a computer PC. The valves V1, V2, V3that are used are connected in the interactive state, since the closingmember thereof in the interior of the valve is pressed against the valveseal by a spring.

List of Reference Numerals

1-lower part

2-inner chamber lower part

3-lower portion

4-upper portion

5-ledge

6-groove

7-suction shaft

8-suction shaft

9-guide tab

10-guide tab

11-guide tab

12-guide tab

13-guide tab

14-sealing surface lower part

15-groove

16-rubber seal

17-grip recess

18-grip recess

19-grip recess

20-bevelled guide edge lower part

21-bore

22-centring shaft

23-centring shaft

30-cover

31-grip recess

32-grip recess

33-grip recess

34-bevelled guide edge cover

35-ledge cover (sealing surface of the upper filter support)

36-sealing surface cover/lower part

37-recess

38-recess

39-recess

40-recess

41-recess

42-seal cover/lower part

43-seal lower support

44-seal upper support

45-bolt

50-bevel guide tab

51-bevel guide tab

60-spacer sleeve

61-slot

62-bevelled end edge

70-module “vacuum chamber”

71-module “control vacuum chamber”

72-module “electronics system”

73-module “valves”

74-module “RD4 oil pump”

75-module “commanding robot”

80-outlet tip

81-filter element

82-outlet tip

83-filter element

84-side wall filter support

V-vacuum chamber

VO-upper vacuum region

VU-lower vacuum region

F1-upper filter support

F2-lower filter support

V1-valve

V2-valve

V3-valve

VF-vacuum trap

RD4-oil pump

PR-pipetting robot

RS-shelving system

K-carousel

GR-gripping robot

RW-Risc-Workstation

T-dryer unit

S-interface

PC-control computer

What is claimed is:
 1. A vacuum system for the directed transport of asubstance, having a vacuum chamber (V) which comprises a first permeablemeans (F1) and a second permeable means (F2), which are arranged oneabove the other, and two vacuum regions (VO, VU) an upper vacuum region(VO) being defines by a space between the first permeable means (F1) andthe second permeable means (F2) and a lower vacuum region (VU) beingdefined by a space between the second permeable means (F2) and a base ofthe vacuum chamber, a vacuum pump RD4), a first electronicallycontrolled valve (V1) for the lower vacuum region (VU), a secondelectronically controlled valve (V2) for the upper vacuum region (VO),and a vacuum trap (VF) arranged at the lower vacuum region (VU) forreceiving a waste volume, a third valve (V3), arranged upstream of thefirst electronic valve (V1), for breaking a creeping vacuum in the loservacuum region (VU), wherein: vacuum is generated and established in eachof the upper and the lower vacuum regions (VO, VU) independently of oneanother so that a substance is transported in two steps through thefirst permeable means (F1) and the second permeable means (F2) into thelower vacuum region (VO).
 2. The vacuum system according to claim 1,wherein the vacuum system has only two vacuum regions (VO, VU).
 3. Thevacuum system according to claim 1, wherein the first permeable means(F1) is formed by a filter support.
 4. The vacuum system according toclaim 3, wherein the second permeable means (F2) is formed by a filtersupport.
 5. The vacuum system according to claim 1, wherein the vacuumsystem has a cover (30) and a lower part (1).
 6. The vacuum systemaccording to claim 5 wherein the lower part (1) of the vacuum chamber(V) has a ledge (5) for supporting the second permeable means (F2). 7.The vacuum system according to claim 5 the lower part includes sidewalls which are provided in the side walls of the lower part (1) of thevacuum chamber (V) recesses (17, 18, 19) for a gripper of a robot (GR).8. The vacuum system according to claim 7, wherein the lower part (1) ofthe vacuum chamber (V) has guide tabs (9, 10, 11, 12, 13) havingcorrecting levels (50, 51) for the insertion of the first permeablemeans (F1) and the second permeable means (F2) into the lower part (1).9. The vacuum according to claim 8, wherein the guide tabs (9, 10, 11,12, 13) have a first and second bevel angle (50, 51).
 10. The vacuumsystem according to claim 9, wherein the first bevel angle (50) is about30° and the second bevel angle (51) is about 2°.
 11. The vacuum systemaccording to claim 5, wherein the lower part (1) has a beveledperipheral guide edge (2), with which the first and second permeablemeans (F1, F2) come into contact on insertion into the lower part (1) ofthe vacuum chamber (V).
 12. The vacuum system according to claim 5,wherein the cover (30) of the vacuum chamber (V) has a beveled guideedge (34), which comes into contact with the first permeable means (F1)when the cover (30), is put in place on the lower part (1).
 13. Thevacuum system according to claim 12, wherein the bevel guide edge is atan angle of 30°.
 14. The vacuum system according to claim 5, wherein thecover (30) has wall region recesses (31, 32, 33) for a gripper of arobot (GR).
 15. The vacuum system according to claim 5, wherein thecover (30) has a supporting surface (35) for sealing the upper filtersupport (F1).
 16. The vacuum system according to claim 15, wherein thesealing materials for the supporting surface sealing the upper filtersupport (F1) has a hardness of 20 Shore.
 17. The vacuum system accordingto claim 16, wherein a joint between the cover (30) and the lower part(1) is formed by a combination of an O-ring (16) and a non-resilientsealing strip (42), the O-ring (16) providing a seal of 60 Shore and thesealing strip (42) of 30 Shore.
 18. The vacuum system according to claim17, wherein the lower filter support (F2) sealing is effected by a seal(43) having a hardness of 60 Shore.
 19. The vacuum system according toclaim 8, wherein the cover (30) has corresponding receiving means (37,38, 39, 40, 41) for receiving the guide tabs.
 20. The vacuum systemaccording to claim 1, wherein the upper filter support (F1) has a numberN of pipe-shaped filter elements that are connected to form a filtersupport.
 21. The vacuum system according to claim 20, wherein the lowerfilter support (F2) has N pipe-shaped filter elements that correspond tothe filter elements (81, 83) of the upper filter support (F1) and areconnected to form a filter support.
 22. The vacuum system according toclaim 21, wherein there are mounted four corner pipes of the two filtersupports (F1, F2) and a spacer sleeve (60) which, in addition to itsfunction of defining the spacing between the two filter supports (F1,F2), also effects a vertical correction of misplacements of the lowersupport (F2) by engaging in centering shafts (22, 23) in the lower part(1) of the vacuum chamber (V).
 23. The vacuum system according to claim22, wherein the spacer sleeves (60) are cylindrical in shape, having athrough slot (61) in the axial direction so that vacuum is able to acton the corner pipes.
 24. The vacuum system according to claim 23,wherein the spacer sleeves (60) have code end faces which are beveled.25. The vacuum system according to claim 22, wherein the length of thespacer sleeves (60) is such that outlet tips (82) of the upper filtersupport (F1) are located inside the pipes (81) of the lower filtersupport (F2).
 26. The vacuum system according to claim 25, wherein theoutlet tips (82) of the upper filter support (F1) are located 1.5 mminside the pipes (81) of the lower support (F2).
 27. The vacuum systemaccording to claim 22, wherein the spacer sleeves (60) are made ofplexiglass.
 28. The vacuum system according to claim 1, wherein thevacuum chamber (V) is manufactured from plexiglass, glass or specialsteel of a suitable thickness.
 29. The vacuum system according to claim28, wherein the vacuum chamber (V) is manufactured by aninjection-moulding process or by a milling process.
 30. The vacuumsystem according to claim 1, wherein the lower part (1) of the vacuumchamber (V) has a suction shaft (7) for the lower vacuum region (VU) anda suction shaft (8) for the upper vacuum region (VO).
 31. The vacuumsystem according to claim 1, wherein each valve (V1, V2, V3) has its owncontrolling electronics system which can be actuated by the controlsoftware via a decoding apparatus of a PC.
 32. The vacuum systemaccording to claim 1, wherein a dry unit (T) for drying the filtersupports (F1, F2) is also provided.
 33. The vacuum system according toclaim 1 further comprising means for transporting a substance through atleast one permeable means (F1) to the second permeable means (F2).